The estrogen receptor (ER) is a ligand-activated transcriptional regulatory factor which modulates the expression of numerous genes. The loss of ER expression in approximately 30% of primary tumors is associated with a more aggressive phenotype, and disallows the use of endocrine therapy. We hypothesize that negative regulation of the ER gene is a major factor contributing to an ER-negative phenotype. This negative regulation may be caused by genomic alterations in the 5' -upstream regulatory region (5'-URR) of the ER gene, or altered levels of transcription regulatory factors which modulate ER gene expression. In Specific Aim 1 we will identify transcriptional regulatory elements contained within the 5'-URR of the ER gene. CAT assays will be used to characterize the activity of promoter/enhancer or suppressor elements contained within the ER 5' -URR in ER-positive and ER-negative breast cancer cells. Potential genomic alterations within the ER 5' -URR will be identified in Specific Aim II by sequencing this region in an ER- negative breast cancer cell-line and ER-negative primary tumors. The functional significance of identified mutations will be assessed by CAT assay. Key factors which modulate ER levels will be characterized in terms of their transcriptional regulatory potential in Specific Aim III. ER-positive breast cancer cells will be treated with these factors, and Northern blot analysis as well as Actinomycin D and nuclear run-on assays will be employed to define those factors which transcriptionally modulate expression of the ER gene. Finally, the effects of these transcriptional modulatory factors on ER promoter/enhancer or suppressor activity will be examined in ER-positive and ER-negative cell-lines by CAT assay.